Granulocytic leukemia cell lines have been demonstrated by us to evolve in vitro in continuous marrow cultures derived from serially transplanted marrow of irradiated RfM but not C3H/HeJ mice. We now propose to quantitate the effects of pluripotentail hematopoietic stem cell proliferative history, committment to granulocyte-macrophage progenitor cells and the role of contact with stromal cells on x-ray/alkylating agent induced granulocytic leukemogenesis in vitro. Cloned permanent normal-diploid, nonleukemogenic, suspension linies of growth factor dependent granulocyte-macrophage progenitor cells, and multipotential hematopoietic stem cells (forming erythroid, mast cell/basophil and granulocyte-macrophage colonies by not CFUs) have been derived in our laboratory from long-term bone marrow cultures of RfM/UN mice (high granulocytic leukemia incidence), C3H/HeJ and C57BL/6J mice (low granulocytic leukemia incidence strains). Corticosteroid supplemented long-term whole vone marrow cultures frm each strain generate pluirpotential stemm cells (CFUs) as well as multipotential and committed stem clles for 20-30 weeks on a stromal cell layer. Purified bone marrow stromal cultures from each strain prepared by growth of whole marrow over 28 days with corticosteroid-deprivation contain no hemopoietic cells. Cloned factor-dependent lines carried in suspension culture or in contact with purified stromal cells and continuous whole marrow cultures frm each strain will be treated in vitro with L-phenylalanene mustrad, x-irradiation (delivered by external beam or by addition of tritiated water to the medium) or both drug and x-ray. Hemopoietic cells will then be serially tested for differentiation capacity, induction of hypoxanthine-guanine phosporbosyl transferase deficiency mutants, and factor-independent clonal sublines producing granulocytic leukemia in vivo. Malignant transformation of factor-dependent preogenitor cell lines will be quantitated during drug/x-ray exposure in suspension culture and during contact with purified stroma. Methods will include continuous bone marrow cultures, clonagenic assays for factor-dependent and independent colony formation in semi-solid medium, CFUs assay, and assay of leukomogenicity in vivo. These data should help to elucidate the mechanism of human cancer treatment related acute myelogenous leukemia.